Example Project 1: Advancing the manufacturing of extracellular vesicles derived from human stem cells for cardiac regenerative medicine.
Foreign Advisor: Dr. Margarida Serra (iBET)
Background: The interest in cell-derived extracellular vesicles (EV) to address unmet medical needs in cardiovascular disease is unquestionable, but the road to its full exploitation depends on the ability to overcome several hurdles in their manufacturing. This project aims at advancing the research and manufacture of EV for cardiac regenerative medicine by generating knowledge on how the source of parent cells influences their secretome as well on the modulation of critical process parameters to intensify their production in stirred-tank bioreactors.
Research Problem: Understanding the impact of parental cell (e.g., mesenchymal stem cell or human induced pluripotent stem cells) and critical process parameters (e.g. stirring-rate, dissolved oxygen, operation mode) on the yield and quality attributes of EVs.
Role of Student in Research: Participate in the development of an optimized bioprocess for culture of parental cells in a 3D culture setting (microcarriers or cell spheroids) in stirred-tank bioreactors. The student will also participate in the characterization of the generated EV.
Equipment/Software Student Will Use/Learn: stirred-tank bioreactors, laminar flow hoods, cell counters, microscopes, NTA, CEDEX Bio, among others.
Example Project 2: Human extracellular-matrix functionalization of 3D hiPSC-based cardiac tissues to improve cardiomyocyte maturation.
Foreign Advisor: Dr. Margarida Serra (iBET)
Background: Human induced pluripotent stem cells (hiPSC) possess significant therapeutic potential due to their high self-renewal capability and potential to differentiate into specialized cells such as cardiomyocytes. However, generated hiPSC-derived cardiomyocytes (hiPSC-CM) are still immature, with phenotypic and functional features resembling the fetal rather than their adult counterparts, which limits their application in cell-based therapies, in vitro cardiac disease modeling, and drug cardiotoxicity screening. Within this context, this project aims to develop efficient strategies to improve hiPSC-CM maturation through the use of extracellular matrix (ECM) derived from human (stem) cells.
Research Problem: Understanding the impact of human cardiac fibroblasts derived ECM in hiPSC-CM maturation.
Role of Student in Research: Participate in the culture and differentiation of hiPSC into cardiac derivatives, decellularization of cell cultures and hydrogel preparation and characterization of hiPSC and cardiac derivatives at different stages.
Equipment/Software Student will Use/Learn: stirred-tank bioreactors, laminar flow hoods, cell counters, microscopes, CEDEX Bio, flow cytometers, among others.
Example Project 3: Development of complex 3D co-culture systems with intact tumor microenvironment.
Foreign advisor: Dr. Catarina Brito (iBET)
Background: Diffuse Midline Glioma (DMG) is a high-grade Glioma (HGG) of the CNS which is localized in the brain pons. DMGs are the most common brainstem gliomas in children, with a 5-year survival rate of 1%. These tumors are characterized by a non-inflammatory “cold” immune tumor microenvironment (TME) which is an obstacle for immunotherapeutic approach effectiveness. Such “cold” immune TME is mostly driven by molecular crosstalk between myeloid cells (resident microglia and infiltrating macrophages), astrocytes, and cancer cells. Still, the molecular targets to counteract such non-inflammatory TME and immunotherapy resistance have not been well defined in DMG due to the lack of research models with human relevance.
Research Problem: Development of a 3D co-culture strategy to integrate the main cell players of the DGM TME, employing controlled stirred-tank bioreactors. The strategy includes: 1) co-differentiation of the main CNS neural cell types (neurons, astrocytes, and oligodendrocytes) and microglia, and 2) the co-culture with blood-derived monocytes and 3D DMG cell cultures.
Equipment/Software Student Will Use/Learn: Animal cell culture systems (e.g., spinner vessels, computer controlled stirred-tank bioreactors), methods for cell monitoring (e.g., fluorescence widefield microscopy), image analysis software (ImageJ).
Role of Student in Research: Optimize culture parameters including dissolved oxygen, cell ratios, agitation rate and cell density towards optimal mass transfer. Monitoring of cell aggregation by spheroid diameter, spheroid morphology, cell viability and phenotype.
Example Project 4: Miniaturization of hepatic cell culture bioprocessing with innate immune function for drug discovery.
Foreign advisor: Dr. Catarina Brito (iBET)
Background: The liver is a central organ, exposed to virtually all blood-borne pathogens and therapeutics (orally and systemically administered). Hepatocytes are responsible for drug metabolism and together with other liver cells (e.g., Kupffer cells, the resident macrophages), elicit early immune responses (e.g., to gene therapy vectors). Dr. Brito has been exploring stirred-tank culture systems (scales 30-200 mL) to develop 3D in vitro models of the liver with innate immune function for drug discovery.
Research Problem: Down-scale the bioprocess for generation of the 3D hepatic models (monocultures and co-cultures) to 15- and 5-mL scales, while maintaining the viability and phenotype of the cells. The miniaturization of the cultures will allow studies with primary biological material, typically limiting (e.g., primary human hepatocytes, primary human macrophages, human infectious pathogens).
Equipment/Software Student Will Use/Learn: Animal cell culture systems (e.g., spinner vessels, controlled stirred-tank bioreactors), methods for cell monitoring (e.g., fluorescence widefield microscopy), image analysis software (ImageJ).
Role of Student in Research: Optimization of agitation rate, cell density, culture volume of 3D mono- and co-culture parameters in different stirred-tank culture systems.
Example Project 5: Unveiling the anticancer potential of a phenolic compound from virgin olive oil in colorectal cancer cells: impact of hydroxytyrosol on cancer cell metabolism.
Foreign Advisor: Dr Teresa Serra (iBET)
Background: Portugal is the world’s 8th largest producer of olive oil. The health benefits of virgin olive oil (VOO) have been widely reported, but the scientific evidence of the effect of this food on colorectal cancer (CRC) is still limited as it is only based on i) epidemiological data (showing a lower risk of CRC incidence with VOO intake) and ii) cell-based studies using monolayers cultures. This proposal aims at generating knowledge on the mechanisms of action of hydroxytyrosol, the main phenolic compound of VOO, in CRC by identifying the metabolic pathways modulated by this compound using a 3D cell model.
Research Problem: Understanding the impact of hydroxytyrosol on the metabolism of LoVo cell spheroids.
Role of Student in Research: Participate in the development of the LoVo spheroids in bioreactors and bioassays with hydroxytyrosol; participate in the sample preparation for the metabolomic studies and data analysis.
Equipment/Software Student will Use/Learn: laminar flow hoods, cell counters, microscopes, software packages for data analysis and integration (e.g., MarkerView, MasterView, LibraryView, MetaboAnalyst3.0).
Example Project 6: Mechanisms of anticancer properties of virgin olive oil phenolic compounds in breast cancer metastasis.
Foreign Advisor: Dr Teresa Serra (iBET)
Background: The phenolic compounds from VOO have been widely reported to have antioxidant potential. The PI’s previous work has found that metastasizing cancer cells subject to fluid shear stress (FSS) have elevated generation of reactive oxygen species, which then triggers the epithelial-to-mesenchymal transition (EMT). This cascade of events of adaptation makes the metastasizing cells primed for a higher degree of invasiveness and aggressive characteristics. Therefore, this project aims to understand how hydroxytyrosol, the main phenolic compound of VOO, can reduce EMT in 3D breast cancer cell models.
Research Problem: Understanding the impact of hydroxytyrosol on the EMT gene expression of cells subject to FSS.
Role of Student in Research: Culture breast cancer cells in sublethal FSS-inducing conditions in bioreactors with and without hydroxytyrosol and perform quantitative PCR analysis of EMT genes. The student will also participate in the sample preparation for the metabolomic studies and data analysis.
Equipment/Software Student will Use/Learn: laminar flow hoods, cell counters, microscopes, quantitative PCR, software packages for data analysis and integration (e.g. MarkerView, MasterView, LibraryView, MetaboAnalyst3.0).